BackgroundOur previous study determined that tetrandrine (Tet) has an inhibitory effect on the proliferation of human Tenon capsule fibroblasts ( TCFs ) in vitro,but its mechanism is poorly understood.ObjectiveThis study was to investigate the effect mechanism of Tet on human TCFs.MethodsHuman TCFs were isolated and cultured from scleral tissue of donor using explant technique.The cells were identified by vimentin antibody staining and morphology.The third generation of cells were seeded in the culture plate at the density of 1 × 105 cells/ml.Twenty-four hours after inoculation,the Tet of 1 × 10-5 mol/L was added in the well of culture plate,and the cells cultured only in 1640 medium served as the control group.The apoptosis of the cells was assessed by TUNEL,and the expressions of bax,bel-2,transforming growth factor-β2 (TGF-β2 ) in TCFs were detected using immunochemistry.Results The cultured cells showed the features of the fibroblasts in shape with the positive response for vimentin.A number of TUNEL positive cells were seen in Tet group and no TUNEL positive response was found in control group.The expression levels (A value) of bax,bcl-2 and TGF-~ protein in TCFs were 0.577 ± 0.009,0.430±0.012 and 0.341 ±0.017 in Tet group,and those in control group were 0.320±0.015,0.819±0.021 and 0.624±0.014 respectively,showing statistically significant differences between two groups( t =33.277,-35.356,-28.093,P＜0.01 ).Conclusions Tet suppresses the proliferation of human TCFs through up-regulating the expression of bax and down-regulating the expressions of bcl-2 and TGF-β2 in vitro.

OBJECTIVETo observe the changes of nuclear factor-kappa B (NF-kappaB) in the course of N-methyl-N-nitrosourea (MNU)-induced apoptosis of rat retinal photoreceptor cells and investigate the mechanism of MNU-induced retinal damage.

METHODSA single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats, which were sacrificed at different intervals after MNU treatment. The retinal damage was examined with optical microscopy and photoreceptor cell apoptosis detected by TUNEL assay. Western blotting was performed to analyze the changes in NF-kappaB.

RESULTSPyknosis of the photoreceptor cell nuclei and disorientation of the outer segment of the photoreceptor layer was observed 24 h after MNU treatment, and the outer nuclear layer and photoreceptor layer were almost completely lost on day 7. Photoreceptor cell apoptosis peaked at 24 h, and in the apoptotic cascade, NF-kappaB p65 protein was only detected 12 and 24 h after MNU treatment, whereas the amount of I kappa B alpha, in contrast, markedly increased in the cytoplasm as well as in the nuclei.

CONCLUSIONMNU-induced retinal damage might be mediated through the signaling pathway of NF-kappaB/I kappa B alpha.

Animals ; Apoptosis ; drug effects ; Blotting, Western ; Female ; I-kappa B Proteins ; metabolism ; In Situ Nick-End Labeling ; Methylnitrosourea ; toxicity ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley ; Retinal Diseases ; chemically induced ; metabolism ; pathology

OBJECTIVETo investigate the change of expression level of metastasis suppressor gene Kiss-1 in the colorectal cancer cell line SW480 after radiation, and to determine its association with the proliferation and apoptosis of SW480 cells.

METHODSSW480 cells were divided into control group (0 Gy) and study groups (2, 4, 6, 8 Gy). Cells in the study groups were irradiated by 6-MV X-ray radiation for 48 hours. Immunohistochemistry and real-time PCR methods were used to investigate the influence of radiation on Kiss-1 gene expression of SW480. Colony formation assay was used to detect the proliferation of SW480. Flow cytometry-Annexin- V/PI assay was used to observe the change of the apoptosis rate.

RESULTSCompared with the control group, Kiss-1 protein expression increased after radiation of 6, 8 Gy (P<0.05), but no significant changes were observed after radiation of 2, 4 Gy(P>0.05). Kiss-1 gene mRNA level increased after radiation of 2, 4, 6 Gy, while no obvious change was observed for 8 Gy radiation. The apoptosis rates increased for 4, 6, 8 Gy radiation(P<0.05), however, there was no significant difference for 2 Gy radiation (P<0.05).

CONCLUSIONRadiation may increase Kiss-1 gene expression in SW480 cells, which results in decreases proliferation and increases apoptosis in residual surviving cells.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Humans ; Kisspeptins ; genetics ; metabolism ; radiation effects ; RNA, Messenger ; genetics ; X-Rays

OBJECTIVETo study the prevention & treatment effect of cordyceps sinensi on bleomycin-induced pulmonary fibrosis (PF) in mice.

METHODRats were divided into 4 groups. In BM group (model), pulmonary interstitial fibrosis model was established through nasal dripping of 5 mg x kg(-1) bleomycin. In PCT group (prevention & treatment with cordyceps sinensi), cordyceps sinensi was orally administered 48 h before bleomycin dripping. In CT group (treatment with cordyceps sinensi), cordyceps sinensi was orally administered at 14 d after bleomycin dripping, and NC group (normal mice, control). At 28 d, bronchoalveolar lavage fluid (BALF) and lung tissue were collected, right lung received HE staining and masson staining, the change in cell micro-structure was observed under electron microscope, the hydroxyproline (HYP) content in lung tissue of left lung was detected through acidolysis method, and TGF-beta1, IFN-gamma and IL-4 concentration in BALF was detected through ELISA.

RESULTCompared with those in BM group, mouse lung coefficient decreased significantly (P < 0.05), alveolitis and fibrosis degree improved significantly in PCT and CT groups (P < 0.05), HYP content in lung decreased (P < 0.05). TGF-beta1 and 1L-4 content in BALF decreased significantly in each drug intervention group, IFN-gamma content increased significantly (P < 0.05).

CONCLUSIONThe cordyceps sinensi can alleviate bleomycin-induced alveolitis and fibrosis degree in a certain degree. The mechanism of the alleviation is possibly due to its inhibition of TGF-beta1 expression and its regulation of imbalance of the type I and II cytokine in lung tissue.

Animals ; Bleomycin ; Bronchoalveolar Lavage Fluid ; chemistry ; Cordyceps ; chemistry ; Hydroxyproline ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Lung ; metabolism ; pathology ; ultrastructure ; Male ; Mice ; Mice, Inbred ICR ; Pulmonary Fibrosis ; chemically induced ; metabolism ; pathology ; Random Allocation ; Transforming Growth Factor beta1 ; metabolism

BACKGROUNDPrevious studies have showed that photooxidative stress can lead to down-modulation of nuclear factor-kappa B (NF-kappaB) activity causing apoptosis of cultured photoreceptor cells. This study aimed at investigating whether NF-kappaB was involved in photoreceptor cells apoptosis induced by N-methyl-N-nitrosourea (MNU) in rats.

METHODSA single intraperitoneal injection of 60 mg/kg MNU was given to 50-day-old female rats. At different intervals after MNU treatment, the animals were sacrificed. Retinal damage was examined by a light microscope. The apoptotic index of the photoreceptor cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). NF-kappaB was analysed by Western blot and Transcriptin Factor Assay Kits.

RESULTSThe pyknosis of the photoreceptor nuclei and the disorientation of the outer segment of the photoreceptor layer was seen after MNU treatment for 24 hours. The outer nuclear layer and photoreceptor layer were almost completely lost at 7 days. Photoreceptor cells apoptosis reached the peaked value at 24 hours. In apoptotic cascade, the protein levels of NF-kappaB p65 were only detected after MNU treatment for 12 and 24 hours in the nucleus. Conversely, the amounts of IkappaBalpha were markedly increased in the cytoplasm as well as in the nucleus. The activity of NF-kappaB p65 in the nucleus was down-modulated in the end.

CONCLUSIONSMNU-induced photoreceptor cell destruction was attributed to the apoptotic process by down-regulating the activation of NF-kappaB p65.

Animals ; Apoptosis ; drug effects ; Cell Nucleus ; metabolism ; Female ; I-kappa B Proteins ; analysis ; physiology ; Methylnitrosourea ; toxicity ; NF-KappaB Inhibitor alpha ; NF-kappa B ; analysis ; physiology ; Photoreceptor Cells ; chemistry ; drug effects ; pathology ; Rats ; Rats, Sprague-Dawley ; Retina ; drug effects ; pathology

OBJECTIVETo investigate whether the phosphorylation (functionally inhibitive) of eukaryotic initiation factor 2-alpha (eIF2-a) affects the molecular mechanism of cisplatin-induced cellular apoptosis in human hepatocellular carcinoma (HCC).

METHODSThe human HCC cultured cell lines SMMC-7221 and HepG2 were treated with cisplatin alone (controls; 24 h) or in combination with pre-transfection of a dominant-negative eIF2-a mutant (eIF2aS51A) or pre-exposure to an eIF2-a-specific phosphatase inhibitor (salubrinal) to decrease or increase the phosphorylation level, respectively. Changes in expression of apoptosis markers were quantitatively and qualitatively assessed by flow cytometry and western blot analysis. The significance of differences among groups was assessed by analysis of variance testing and of differences between groups was assessed by t-test.

RESULTSCisplatin treatment induced the appropriate functional-inhibitive phosphorylation of eIF2-a on serine 51. Cisplatin treatment (10 mg/ml) induced significant apoptosis in the eIF2aS51A pre-transfected SMMC-7721 (control: 21.7 +/- 1.5% vs. 50.7 +/- 2.1%, t = 19.454, P less than 0.05) and HepG2 (21.0 +/- 1.0% vs. 57.3 +/- 2.1%, t = 27.250, P less than 0.05). Salubrinal pre-treatment significantly inhibited the cisplatin (15 mg/ml)-induced apoptosis in SMMC-7721 (control: 50.3 +/- 2.5% vs. 16.3 +/- 2.1%, t = 18.031, P less than 0.05) and HepG2 (42.0 +/- 2.6% vs. 12.0 +/- 2.0%, t = 15.667, P less than 0.05).

CONCLUSIONPhosphorylation of eIF2-a may act to inhibit cisplatin-induced apoptosis of HCC.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Eukaryotic Initiation Factor-2 ; metabolism ; Humans ; Liver Neoplasms ; Phosphorylation

OBJECTIVETo investigate the cross-talk between the PI3K/Akt and MEK/ERK pathways and its role in cell cycle regulation under endoplasmic reticulum stress in human hepatocellular carcinoma cells.

METHODSPI3K inhibitor LY294002 and MEK inhibitor U0126 were used to block the PI3K/Akt and MEK/ERK pathways respectively, and constitutively activated Akt mutant construct was used to activate the PI3K/Akt pathway. Western blot was used to study the potential cross-talk between the PI3K/Akt and MEK/ERK pathways under endoplasmic reticulum stress in human hepatocellular carcinoma cells. the role of the cross-talk between the PI3K/Akt and MEK/ERK pathways in cell cycle regulation was investigated by using propidium iodide staining.

RESULTSLY294002 not only blocked Akt activation efficiently but also increased ERK phosphorylation markedly under endoplasmic reticulum stress in SMMC-7721 and Hep3B cells. Furthermore, myr-Akt inhibited endoplasmic reticulum stress-mediated ERK phosphorylation. In contrast, MEK inhibitor U0126 had no effect on endoplasmic reticulum stress-induced Akt activation. It is notable that both myr-Akt overexpression and MEK inhibitor U0126 inhibited endoplasmic reticulum stress-induced G0/G1 phase arrest in SMMC-7721 cells.

CONCLUSIONEndoplasmic reticulum stress-induced Akt activation is mediated through PI3K and the PI3K/Akt pathway inactivation is involved in increased ERK activity in human hepatocellular carcinoma cells. The cross-talk between the PI3K/Akt and MEK/ERK cascades plays an important role in endoplasmic reticulum stress-induced human hepatocellular carcinoma cell cycle arrest.

Butadienes ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Chromones ; pharmacology ; Endoplasmic Reticulum ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Morpholines ; pharmacology ; Nitriles ; pharmacology ; Phosphatidylinositol 3-Kinase ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; metabolism ; Signal Transduction

Objective To analyze the imaging and pathological characteristics, as well as treatment strategies of intracranial hemangioblastomas (HBs),and explore the advancement of diagnosis,etiopathogenisis and treatment of HBs. Methods Thirty-five patients with intracranial HBs,admitted to our hospital and performed tumor resection from January 2005 to January 2010,were chosen in our study; all patients were divided into type of big cystic HBs with a small mural nodule (n=19),type of small cystic HBs with a big nodule (n=9) and type of solid HBs (n=7) by imaging features.The clinical manifestations,imaging findings and surgical methods were retrospectively analyzed; the expressions of NSE and CD34 in these tumor samples were detected by HE staining and immunohistochemical staining.Results All patients were treated by surgery; total resection was achieved in 34 and subtotal resection in 1; no death occurred after the surgery.Twenty-eight patients were followed up for 3 months to 3 years after discharge; recurrence appeared in 1 patient with big cystic HBs with a small mural nodule and Gamma knife treatment was performed.No significant difference was observed in the numbers ofCD34+cells between each 2 types of patients (P＞0.05).The numbers of NSE positive cells between each 2 types were statistically significant (P＜0.05). Conclusion There were no specific clinical manifestations of HBs.Diagnosis was mainly according to imaging features.Treatment of HBs with total resection is just the first selection and the key to reduce palindromia; the formation of HBs cysts is closely related to tumor stromal cells.

OBJECTIVETo study the specific cytotoxicity mediated by anti-P-gp/anti-CD(3) diabodies in multidrug resistant solid tumor using P-gp as target.

METHODSThe anti-P-gp/anti-CD(3) diabodies were secreted from E. coli strain 16C9 containing the expression plasmid PAYZDCP, grown at 30 degrees C in a shaker flask; the diabodies were purified by affinity chromatography and identified by SDS-PAGE; the effect of the anti-P-gp/anti-CD(3) diabody mediated lysis of P-gp-expressing tumor cells was assayed by (51)Cr release assay in vitro, and by human KB nude mouse xenograft models in vivo.

RESULTSThe diabodies were generated by bacteria as a soluble functional form and purified by one-step affinity chromatography with a yield > 4 mg/L culture medium. In (51)Cr release assay, the diabodies targeted human activated T cells to lyse P-gp(+)-KB/MDR cells in a dose-dependent manner. It suggested that the diabody was able to induce an efficient lysis of the target cells by human T cells in vitro. When combined with activated human T cells, the diabody significantly inhibited the growth of KB/MDR, but had no effect on KB xenografts.

CONCLUSIONThe anti-P-gp/anti-CD(3) bispecific antibody is a potent agent for targeting human T lymphocytes to lyse solid tumor cells overexpressing P-gp in vitro and in vivo.

ATP-Binding Cassette, Sub-Family B, Member 1 ; immunology ; Animals ; Antibodies, Bispecific ; immunology ; therapeutic use ; CD3 Complex ; immunology ; Drug Resistance, Multiple ; immunology ; Drug Resistance, Neoplasm ; immunology ; Female ; Humans ; KB Cells ; Mice ; Mice, Nude ; Neoplasms, Experimental ; therapy ; Protein Engineering ; methods ; Recombinant Proteins ; therapeutic use ; T-Lymphocytes, Cytotoxic ; immunology

The use of tumor antigen specific antibodies for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. However, several factors restrict the use of anti-PGP monoclonal antibodies(Mabs). First, Pgp is expressed in normal tissues, particularly in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroids plexus and other organs. It plays a significant role to transport drugs and toxins in these organs. Therefore, anti-PGP antibodies in combination with cytotoxic compounds or radiolabelled antibodies should neither inhibit the activity of PGP, nor harm the cells which expressed PGP normally. BiMab exploit the specificity of Mab and ensures activation of cellular cytotoxic mechanisms which kill tumor cells only, but not harm normal cells. It will provide a strategy for resistant cancer therapy using anti-PGP antibodies. Second, Repeated administration of murine antibodies generates a strong human anti-mouse immune (HAMA) response in up to 50% of patients after the first dose, and appro ximately 90% following a second treatment. In an effort to reduce the toxicity and antigenicity, we focus to produce anti-PGP antibodies which have the binding activity only, but not inhibit the function of the "pump", and to construct a small and partially humanized recombinant molecule with dual specificity for both PGP and CD3 complex to activate the host immune response toward the tumour. PCR and overlap PCR were used to construct anti-CD3/ anti-Pgp Diabody. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by both the detection of western blot and size exclusion chromatography; its antigen-binding activity was examined by FACS, cellular RIA. Plasmid pAYZDCP which expressed the anti-CD3/anti-Pgp Diabody was constructed correctly. The diabody was recovered in high yield( up to 2mg/ L) after E-taq purification and predominantly(90%) as a dimer. The diabody can bind to Jurkat cells (CD3+) and K562/A02 cells(Pgp+). The affinities of the diabody were similar with the anti-CD3 ScFv or anti-Pgp ScFv, respectively. The anti-CD3/ anti-Pgp BsF(ab')2 was first recast into the diabody format and succeeded to obtain high level expression. The results of some biological activity experiments indicated that the diabody could bind to Jurkat cells and K562/A02 cells. Multidrug resistance can be reversed experimentally by a variety of drugs, among which the best known are verapamil and trifluoperazine, which unfortunately are of limited use in practice due to severe collateral cardiac toxicity. Anti-PGP x anti-CD3 diabody will provide another therapeutic strategy against multidrug resistance cancer.
ATP-Binding Cassette, Sub-Family B, Member 1 ; immunology ; Animals ; Antibodies, Monoclonal ; immunology ; metabolism ; Antibody Specificity ; genetics ; physiology ; Blotting, Western ; CD3 Complex ; immunology ; Chromatography, Gel ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Flow Cytometry ; Humans ; Jurkat Cells ; drug effects ; metabolism ; K562 Cells ; drug effects ; metabolism ; Mice ; Polymerase Chain Reaction ; Radioimmunoassay ; Trifluoperazine ; pharmacology ; Verapamil ; pharmacology